Transducer for microfluid handling system

ABSTRACT

The present invention relates to integrated micro-cantilevers, micro-bridges or micro-membranes in micro-liquid handling systems. Such micro-liquid handling systems provide novel detection mechanisms for monitoring the physical, chemical and biological properties of fluids in such systems. The present invention further relates to micro-cantilever, micro-bridge or micro-membrane type sensors having integrated readout. Such constructions allow laminated flows of different liquids to flow in a channel without mixing, which opens up for new type of experiments and which reduces noise related to the liquid movement. The present invention even further relates to sensors having adjacent or very closely spaced micro-cantilevers, micro-bridges or micro-membranes which can be exposed to different chemical environments at the same time.

FIELD OF THE INVENTION

[0001] The present invention relates to a sensor using microscopicflexible mechanical structures such as micro-cantilevers, micro-bridgesor micro-membranes integrated into microscopic chambers. In particular,the present invention relates to a sensor for measuring biochemicalproperties of fluids in such chambers.

TECHNICAL BACKGROUND

[0002] The measurement of the properties of fluids flowing inmicroscopic channels is of importance in the field of micro liquidhandling systems, which includes systems for measuring:

[0003] 1) physical properties such as flow rates viscosity and localtemperature

[0004] 2) chemical properties such as pH and chemical composition

[0005] 3) biological properties such as identification of organicconstituents in fluids, including DNA fragments, proteins, and completebiological cells

[0006] Microliquid handling systems typically consist of narrow channelsof order 100 microns wide and 100 microns deep engraved or embossed intothe surface of a thin wafer of a material such as silicon, glass orplastic using reproduction techniques based on micromachining. Thesurface containing the channels is usually bonded to another surface, inorder to seal the channels. Fluids pumped through the resulting channelstypically flow in a completely laminar fashion. As a result, severaldifferent fluids can be flowed in laminated streams through suchMicrosystems, without any significant mixing of the fluids.

[0007] An important advantage of a microliquid handling system is thatvery small quantities of fluid can be directed in a controlled fashionto various parts of the system, where various analytical techniques canbe used to determine the properties of the liquid. This can be doneusing external analytical techniques such as optical detection. Thecontrolled flow of the fluid is achieved via pumps and valve systemsthat can be either external or integrated with the microchannels.

[0008] Micro-cantilevers are devices where changes in the mechanicalproperties of a microscopic micro-cantilever are used to detect changesin the environment of the micro-cantilever. The micro-cantilever istypically of the order of 100 microns long, 10 microns wide and onemicron thick. The micro-cantilevers are made of a material such assilicon, silicon nitride, glass, metal or combination of any of these,using micromachining techniques. A change in the mechanical propertiescan for example be a stress formation in the micro-cantilever due tochanges in surface stress of the micro-cantilever. Stress formation canalso occur due to changes in temperature of the micro-cantilever due toa bimorph effect, if the micro-cantilever is made of two materials withdifferent thermal expansion coefficients. Such stress formations in themicro-cantilever can be detected in a variety of ways. Often the stressformation will result in a deflection of the micro-cantilever. In thesesituations the deflection can be detected by deflection of a laser lightbeam by a reflecting surface of the micro-cantilever. Change in theresistivity of a piezoresistor integrated onto the micro-cantilever isanother method, which has the advantage that it does not depend on adeflection of the micro-cantilever and it does not require opticalaccess to the micro-cantilever.

[0009] Change in resonance frequency is another example of a change in amechanical property. A change in mass of the micro-cantilever can occurif material binds to the micro-cantilever, and such a change willproduce a change in the resonance frequency of the micro-cantilever.Such changes can be monitored by actuating the micro-cantilever at afrequency near its resonance frequency, and monitoring changes in theamplitude of the resulting dynamic bending of the micro-cantilever,using methods similar to those described above for the detection ofstress formation.

[0010] Using these changes in mechanical properties, micro-cantilevers,have been used to detect chemical reactions occurring on the surface ofthe micro-cantilever, either in gas phase or in liquid phase. For gasphase experiments the measurements have been performed in a gas chamberutilizing optical detection of a micro-cantilever bending.Micro-cantilevers with integrated piezoresistive read-out have been usedfor thermogravimetry in air. Under ambient conditions themicro-cantilever-based detection technique has proven very sensitive. Ithas been demonstrated that mass changes down to 0.5 ng and temperaturechanges down to approximately 10⁻⁵ C. can be resolved. Furthermore, achange of surface stress on the order of 10⁻⁵ N/m has been detected. Inliquids, J. Chen [J. Chen, Ph. D thesis Simon Fraiser University (1995)]reports on a piezoresistive micro-cantilever for mass change detection.Detection of polystyrene spheres was performed in a 3 water tank inwhich the micro-cantilever was placed. By vibrating themicro-cantilever, changes in the resonance frequency and thereby masschanges of the micro-cantilever could be monitored. The micro-cantileverdeflection was monitored by integrated piezoresistive read-out.

[0011] PCT patent application WO99/38007 published Jul. 29, 1999describes a system for detecting analytes in a fluid usingfunctionalised micro-cantilevers mounted in a tube. A bending of themicro-cantilever is induced by molecular interactions on one side of themicro-cantilever. The bending is monitored optically by the reflectionof a laser beam of the end of the micro-cantilever. Examples ofapplication include the formation of self assembled monolayers (SAM's)of alkylthiols on a goldcoated micro-cantilever and the partiallyreversible adsorption of low density lipoproteins. The possibility oftesting multiple analytes against multiple analytes is mentioned. Asolution for generating a reference signal is proposed exploiting thetwisting movement of the micro-cantilever and the ability to distinguishthe twisting from the bending movement. Low flow rates are recommendedin order to avoid perturbations of the micro-cantilever. This is a clearindication that the envisioned flow system is of macroscopic dimensions.

[0012] A micro-cantilever array placed at the top of an open channel hasbeen realised in polymer [C. P. Lee et al., Prooceeding of the μTAS'98workshop (1998) 245-252; L. P. Lang et al., Sensors and Actuators A 71(1998) 144-149]. C. P Lee et al. suggest that these micro-cantileverscan be modified for the use of biochemically functionalized tips for usein atomic force microscopy (AFM) or in scanning near field microscopy(SNOM). Hence, this proposed application is related to surface imaging.

[0013] Commercially available micro-cantilevers have been used assensors in liquid. D. R. Baselt et al. [D. R. Baselt et al., Proceedingsof the IEEE. Vol. 85 4 (1997) 672-679] report on piezoresistivemicro-cantilevers applied as biosensors using magnetic particles. Thecoated micro-cantilevers are placed in a liquid cell in which thedetection takes place. The micro-cantilevers measure the interactionbetween particles immobilised on magnetic beads and the immobilisedparticles on the micro-cantilever surface. If the magnetic beads bind tothe surface, the application of a large magnetic field will cause abending of the micro-cantilever.

[0014] U.S. Pat. No. 5,719,324 describes a micro-cantilever basedsensor, where a mass change of the micro-cantilever is detected as achange in the resonance frequency of the micro-cantilever. Furthermore,a stress change of a micro-cantilever material is monitored as amicro-cantilever deflection. For mass detection, a piezoelectricactuator oscillates the micro-cantilever and the micro-cantileverdeflection is registered by optical read-out. It is mentioned that themass detection principle can also be applied in liquid.

[0015] It is a disadvantage of the above-mentioned systems thatmicro-cantilever based experiments are carried out in large liquidcontainers. Such large liquid container systems are very difficult tostabilise thermally. Furthermore, in such large container systems therequired volume of chemicals is unnecessary high.

[0016] It is a further disadvantage of most of the above-mentionedsystems that the micro-cantilever deflection is detected optically. Thisdisadvantage is due to the fact that it may be difficult to obtainoptical access to a specific micro-cantilever—especially in the casewhere a plurality of micro-cantilevers are closely spaced and in thecase where the liquid is not transparent.

[0017] It is an object of the present invention to integratemicro-cantilevers, micro-bridges or micro-membranes into closedmicro-liquid handling systems, in order to provide novel detectionmechanisms for monitoring the physical, chemical and biologicalproperties of fluids in such systems.

[0018] It is a still further object of the present invention to providea micro-cantilever, micro-bridge or micro-membrane type sensor havingintegrated readout. A closed micro-liquid handling system allowslaminated flows of different liquids to flow in the channel withoutmixing, which opens up for new type of experiments and which reducesnoise related to the liquid movement.

[0019] It is a still further object of the present invention to provideadjacent or very closely spaced micro-cantilevers, micro-bridges ormicro-membranes which can be exposed to different chemical environmentsat the same time by:

[0020] Laminating the fluid flow vertically in the micro-channel intotwo or more streams, so that micro-cantilevers or micro-membranes onopposing sides of the micro-channel are immersed in different fluids, orso that a micro-cantilever, micro-bridge, or micro-membrane is exposedto two different fluids.

[0021] Laminating the fluid flow horizontally in the micro-channel, sothat micro-cantilevers or micro-bridges recessed to different levels inthe micro-channel or micro-membranes placed at the top and at the bottomof the channel are exposed to different fluids.

[0022] In this way, changes in viscous drag, surface stress,temperature, or resonance properties of adjacent or closely spacedmicro-cantilevers, micro-bridges or micro-membranes induced by theirdifferent fluid environments, can be compared.

[0023] Neighbouring or very closely spaced micro-cantilevers,micro-bridges or micro-membranes can be coated with different chemicalsubstances using the method just described for immersing adjacent orneighbouring micro-cantilevers, micro-bridges or micro-membranes indifferent fluids. After coating, the micro-channels can be flushed withother fluids to remove the coating material, and to compare thereactivity of neighbouring or very closely spaced micro-cantilevers,micro-bridges or micro-membranes with different coatings.

[0024] It is a still further object of the present invention to providea micro-cantilever, micro-bridge or micro-membrane based sensor wherethe liquid volume is minimised in order to reduce the use of chemicalsand in order to obtain a system which is easy to stabilise thermally.

SUMMARY OF THE INVENTION

[0025] The above-mentioned objects are complied with by providing, in afirst aspect, a sensor for detecting the presence of a substance in afluid, said sensor comprising:

[0026] means for handling the fluid, said handling means comprising aninteraction chamber of micrometer dimensions, an inlet and an outlet,

[0027] a first flexible member having a surface, said surface holding asubstance, wherein the surface holding the substance is at least partlypositioned inside the interaction chamber so that at least part of thesubstance is exposed to the fluid, and

[0028] means for detecting a mechanical parameter associated with thefirst flexible member, said mechanical parameter being related to thepresence of the substance in the fluid.

[0029] By micrometer dimension is meant that the interaction chamber hasdimensions in the 50-500 microns range (width and depth). The firstflexible member may comprise a micro-cantilever having a first and asecond end, the first end being attached to the interaction chamber. Themicro-cantilever may have a rectangular form and may be approximately 50μm wide, 200 μm long and 1 μm thick.

[0030] The mechanical parameter being associated with the first flexiblemember may both be a static or dynamic parameter. By static is meantthat the flexible member may be subject to a static deformation—e.g.bending. Static deformations are typically induced by stress changes inthe flexible member. By dynamic is meant the flexible member may bedriven at or near its mechanical resonance frequency. Upon detection ofa substance in the fluid the resonance frequency may chance due to achange of mass of the flexible member.

[0031] Alternatively, the first flexible member may comprise amicro-bridge having a first and a second end, wherein the first andsecond ends are attached to the interaction chamber. The dimensions(wide, length and thickness) of a micro-bridge may be similar to thedimensions of the micro-cantilever. Alternatively, the first flexiblemember may form part of a boundary defining the interaction chamber. Theboundary may here be one of the sidewalls of the interaction chamber.

[0032] The detecting means for detecting the mechanical parameterassociated with the first flexible member may comprise a piezoresistiveelement, preferably being an integral part of the first flexible member.Preferably, the piezoresistive element forms part of a balanced bridge,such as a Wheatstone bridge. Alternatively, the detecting means maycomprise a laser, an optical element and a position sensitive photodetector.

[0033] The sensor according to the first aspect of the present inventionmay further comprise an actuator for moving the flexible member relativeto the interaction chamber. The actuator may be implemented in severalways—e.g. by comprising piezoelectric elements, comprising means forproviding an electrostatic induced movement, comprising means forproviding a magnetic induced movement, or by comprising means forproviding a thermal induced movement.

[0034] The handling means may be fabricated in a material selected fromthe group consisting of metals, glasses, polymers or semiconductormaterials, such as silicon.

[0035] The substance being held by the surface of the first flexiblemember may be selected from the group consisting of metals, polymers,biochemical molecules or micro-biochemical structures. The group ofbiochemical molecules and micro-biochemical structures comprisesenzymes, DNA, Cells and proteins.

[0036] The sensor according to the first aspect of the invention mayfurther comprise a second flexible member being at least partlypositioned inside the interaction chamber so that at least part of thesecond flexible member is exposed to the fluid The sensor may furthercomprise means for detecting a mechanical parameter associated with thesecond flexible member. This detecting means may comprise apiezoresistive element being an integral part of the second flexiblemember. The piezoresistive element may form part of a balanced bridge,such as a Wheatstone bridge.

[0037] The second flexible member may serve as a reference to the firstflexible member and thereby being adapted to generate a reference signalvia the detecting means.

[0038] In a second aspect, the present invention relates to a sensor fordetecting the presence of a substance in a fluid, said sensorcomprising:

[0039] means for handling the fluid, said handling means comprising aninteraction chamber, an inlet and an outlet,

[0040] a first flexible member having a surface, said surface holding asubstance, wherein the surface holding the substance is at least partlypositioned inside the interaction chamber so that at least part of thesubstance is exposed to the fluid, and

[0041] means for detecting a mechanical parameter associated with thefirst flexible member, said mechanical parameter being related to thepresence of the substance in the fluid, wherein the detecting means forman integral part of the first flexible member.

[0042] The first flexible member, the detecting means, the actuator maybe implemented as previously mentioned. The interaction chamber may beof micrometer dimensions—i.e. the 50-500 μm range.

[0043] The handling means may be fabricated in a material selected fromthe group consisting of metals, glasses, polymers or semiconductormaterials, such as silicon. The substance being held by the surface ofthe first flexible member may be selected from the group consisting ofmetals, polymers, biochemical molecules or micro-biochemical structures.The group of biochemical molecules and micro-biochemical structurescomprises enzymes, DNA, Cells and proteins.

[0044] In order to obtain a reference signal the sensor according to thesecond aspect may further comprise

[0045] a second flexible member being at least partly positioned insidethe interaction chamber so that at least part of the second flexiblemember is exposed to the fluid, and

[0046] means for detecting a mechanical parameter associated with thesecond flexible member.

[0047] Also here the detecting means may comprise a piezoresistiveelement, said piezoresistive element being an integral part of thesecond flexible member, and wherein the piezoresistive element formspart of a balanced bridge, such as a Wheatstone bridge.

[0048] In a third aspect, the present invention relates to a sensor fordetecting the presence of a substance in a fluid, said sensorcomprising:

[0049] means for handling the fluid, said handling means comprising aninteraction chamber, an inlet and an outlet,

[0050] a first flexible member having a surface, said surface holding asubstance, wherein the surface holding the substance is at least partlypositioned inside the interaction chamber so that at least part of thesubstance is exposed to the fluid, and wherein the first flexible memberforms an integral part of the handling means, and

[0051] means for detecting a mechanical parameter associated with thefirst flexible member, said mechanical parameter being related to thepresence of the substance in the fluid.

[0052] That the flexible member forms an integral part of the handlingalso means that the flexible member may be fabricated separately andthen afterwards being attached to the handling means using a plug-on orsnap-on solution. The handling and flexible member may then afterwardsbe encapsulated to form at least part of the final sensor.

[0053] Again, the first flexible member, the detecting means, and theactuator may be implemented as previously described. Also suitablematerials for fabrication of the handling means and suitable substanceshave previously been described.

[0054] Furthermore, the detecting means for detecting the mechanicalparameter associated with the first flexible member may comprise alaser, an optical element and a position sensitive photo detector.

[0055] A reference signal may be generated by a second flexible memberbeing at least partly positioned inside the interaction chamber so thatat least part of the second flexible member is exposed to the fluid. Thereference signal itself may be generated by a detecting means fordetecting a mechanical parameter associated with the second flexiblemember. The detecting means may comprise a piezoresistive element, saidpiezoresistive element being an integral part of the second flexiblemember, and wherein the piezoresistive element forms part of a balancedbridge, such as a Wheatstone bridge.

[0056] In a fourth aspect, the present invention relates to a sensor fordetecting the presence of a substance in a fluid, said sensorcomprising:

[0057] means for handling the fluid, said handling means comprising aninteraction chamber, an inlet and an outlet,

[0058] a first flexible member having a surface, said surface holding asubstance, wherein the surface holding the substance is at least partlypositioned inside the interaction chamber so that at least part of thesubstance is exposed to the fluid, and wherein fabrication of the firstflexible member is part of fabrication of the handling means,

[0059] means for detecting a mechanical parameter associated with thefirst flexible member, said mechanical parameter being related to thepresence of the substance in the fluid.

[0060] The fact that the fabrication of the first flexible member ispart of the fabrication of the handling means is to be understood in thefollowing way. The fabrication of the handling means involves aplurality of steps. One or more of these step may involve thefabrication of the first flexible member. This issue is addressed infurther details in “Detailed description of the invention”

[0061] The first flexible member, the detecting means, and the actuatormay be implemented as previously described. Also suitable materials forfabrication of the handling means and suitable substances havepreviously been described. Also according to this aspect, the sensor mayfurther comprise

[0062] a second flexible member being at least partly positioned insidethe interaction chamber so that at least part of the second flexiblemember is exposed to the fluid, and

[0063] means for detecting a mechanical parameter associated with thesecond flexible member.

[0064] In a fifth aspect, the present invention relates to a sensor fordetecting the presence of a first and a second substance in a fluid,said sensor comprising:

[0065] means for handling the fluid, said handling means comprising aninteraction chamber of micrometer dimensions, an inlet and an outlet,

[0066] a first flexible member having a surface, said surface holding afirst substance, wherein the surface holding the first substance is atleast partly positioned inside the interaction chamber so that at leastpart of the first substance is exposed to the fluid,

[0067] a second flexible member having a surface, said surface holding asecond substance, wherein the surface holding the second substance is atleast partly positioned inside the interaction chamber so that at leastpart of the second substance is exposed to the fluid,

[0068] a first detecting means for detecting a first mechanicalparameter associated with the first flexible member, said firstmechanical parameter being related to the presence of the firstsubstance in the fluid, and

[0069] a second detecting means for detecting a second mechanicalparameter associated with the second flexible member, said secondmechanical parameter being related to the presence of the secondsubstance in the fluid.

[0070] The first and second flexible members may comprise amicro-cantilever having a first and a second end, wherein the first endis attached to the interaction chamber. Alternatively, the first andsecond flexible members may comprise a micro-bridge having a first and asecond end, wherein the first and second ends are attached to theinteraction chamber. Finally, each of the first and second flexiblemembers may form part of a boundary defining the interaction chamber.This boundary may be a sidewall of the interaction chamber.

[0071] The detecting means may comprise piezoresistive elements beingintegral parts of the first flexible member. The detecting means mayalso comprise lasers,optical elements and a position sensitive photodetectors.

[0072] The sensor may further comprise actuators for the flexiblemembers. These actuators may comprise piezoelectric elements beingintegral parts of the micro-cantilevers. Other types of actuators mayalso be applied.

[0073] The handling means may be fabricated in a material selected fromthe group consisting of metals, glasses, polymers or semiconductormaterials, such as silicon. The substances being held by the surface ofthe first and second flexible members may be selected from the groupconsisting of metals, polymers, biochemical molecules ormicro-biochemical structures. The group of biochemical molecules andmicro-biochemical structures comprises enzymes, DNA, Cells and proteins.

[0074] In a sixth and final aspect, the present invention relates to asensor for detecting the presence of a first and a second substance in amoving laminated fluid, said laminated fluid comprising, in a crosssection perpendicular to a direction of movement, a first and a secondregion, said sensor comprising:

[0075] means for handling the laminated fluid, said handling meanscomprising an interaction chamber, an inlet and an outlet,

[0076] a first flexible member having a surface, said surface holding afirst substance, wherein the surface holding the first substance is atleast partly positioned inside the interaction chamber so that at leastpart of the first substance is exposed to the first region of thelaminated fluid,

[0077] a second flexible member having a surface, said surface holding asecond substance, wherein the surface holding the second substance is atleast partly positioned inside the interaction chamber so that at leastpart of the second substance is exposed to the second region of thelaminated fluid,

[0078] means for detecting a first mechanical parameter associated withthe first flexible member, said first mechanical parameter being relatedto the presence of the first substance in the first region of the fluid,and

[0079] means for detecting a second mechanical parameter associated withthe second flexible member, said second mechanical parameter beingrelated to the presence of the second substance in the second region ofthe fluid.

[0080] By a moving laminated flow is meant that a measurements may beperformed in a continues liquid flow or, alternatively, that the liquidis introduced into the chamber and then temporarily stopped while themeasurements are being performed. After the measurements have beenperformed the liquid is guided away from the chamber.

[0081] The detecting means for detecting the mechanical parametersassociated with the first and second flexible members may comprisepiezoresistive elements being integral parts of the flexible members.Alternatively, the detecting means for detecting the first and secondmechanical parameters associated with the first and second flexiblemember, may comprise lasers, optical elements and a position sensitivephoto detectors.

[0082] Furthermore actuators may be applied for moving part of theflexible elements relative to the handling means. These actuators maycomprise piezoelectric elements, said piezoelectric elements beingintegral parts of the flexible members. Also with regard to this aspect,the handling means may be fabricated in a material selected from thegroup consisting of metals, glasses, polymers or semiconductormaterials, such as silicon. The substances to by held be the flexiblemembers may be as previously mentioned.

[0083] It is an advantage of the present invention that piezoresistorsare integrated and used to measure the deflections of the flexiblemembers.

[0084] It is a still further advantage of the present invention that aplurality flexible members can be integrated closely together in amicro-system, so that one flexible member can serve as a reference toanother, or that nearby flexible members can be immersed in differentlaminated streams in a fluid flow, so that one fluid can serve as areference to another.

[0085] It is a still further advantage of the present invention that itprovides a sensor where the liquid volume is minimised in order toreduce the use of chemicals and in order to obtain a system which iseasy to stabilise thermally.

[0086] The above object, advantages and features, together with numerousother advantages and features will be evident from the detaileddescription below of preferred embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0087]FIG. 1: Schematic cross-sectional view of a micro-channel withintegrated micro-cantilever.

[0088]FIG. 2: Recessed micro-cantilevers placed in a micro-channel.

[0089]FIG. 3: Two micro-cantilever resistors and two support resistorsplaced in a Wheat-stone bridge.

[0090]FIG. 4: Schematic drawing of triangular micro-cantilever with onepiezoresistor placed on each arm.

[0091]FIG. 5: Schematic cross-sectional view of the process sequence ofthe lower part of the channel and the micro-cantilever.

[0092]FIG. 6: Schematic top view of the micro-cantilever-basedbiochemical sensor after the fabrication of the lower part of thechannel

[0093]FIG. 7: Schematic cross-sectional view of the process sequence,with the lower part of the channel defined by RIE and the upper part ofthe channel defined by the use of a photosensitive polymer as spacerlayer

[0094]FIG. 8 Schematic cross-sectional view of the process sequence,with the lower part of the channel defined by wet etching and the upperpart of the channel by use of a photosensitive polymer as spacer layer

[0095]FIG. 9: Schematic cross-sectional view of the process sequence ofthe upper part of the channel. The channel is defined by the use ofanodic bonding.

[0096]FIG. 10: Schematic top view of the micro-membrane-basedbiochemical sensor with the channel made of a photosensitive polymer.

[0097]FIG. 11: Principle of exposing closely spaced micro-cantilevers todifferent chemical environments by using vertically laminated flow (a),horizontally laminated flow (b) and by moving the micro-cantileverthrough different layers of laminated flow (c).

[0098]FIG. 12: Principle of using one micro-cantilever as measurementmicro-cantilever and the other micro-cantilever as reference.

[0099]FIG. 13: Principle of using two laminar flow. One as themeasurement flow and the other as a reference flow.

[0100]FIG. 14: Experimental set-up for the measurement of alcoholdiffusion in a polymer coated micro-cantilever.

[0101]FIG. 15: Micro-cantilever response to injected alcohol as afunction of time.

DETAILED DESCRIPTION OF THE INVENTION

[0102] Micrometer-sized mechanical structures such as micro-cantilevers,micro-bridges and micro-membranes can be used as very sensitive sensorsin environments ranging from cryogenic temperatures and ultra-highvacuum to ambient conditions and physiological liquids. Especially thelatter makes it interesting for biochemical applications.

[0103] Basically, a biochemical reaction at a micro-cantilever,micro-bridge or micro-membrane surface can result in a temperaturechange or in a change in the surface stress. The temperature change isobserved by coating the microscopic flexible structure with a metallayer. As a result, the flexible sensor will be stressed due to thebimetallic effect. Furthermore, a change in mass load can be detected asa change in the resonance frequency of the microscopic flexiblestructure. In order to detect biochemical reactions at the microscopicflexible structure surface, part of the microscopic flexible structuremust be coated with a ‘detector film’ that reacts with the bio-moleculesunder investigation.

[0104] For experiments in liquid it has seen to be crucial to includereference measurements on a flexible structure which has not been coatedwith a detector film. If the coated and uncoated flexible structure areplaced closely together in the same environment, the referencemeasurement can be used to cancel out background noise related to forexample liquid movement and thermal drift.

[0105] By monitoring the stress formation in the microscopic flexiblestructure or the microscopic flexible structures resonance frequency asa function of time it is possible to study the kinetics of surfaceprocesses. One very promising application is to use an array ofmicroscopic flexible structures in order to detect the presence ofdifferent kinds of molecules simultaneously.

[0106] Often, a change in mechanical properties is detected as adeflection of the microscopic flexible structure using an externaloptical system. However, for an array of microscopic flexiblestructures, this type of read-out becomes very complicated and operationin liquid is even more problematic. Moreover, this read-out depends on ameasurable deflection of the microscopic flexible structure. For arrayand liquid applications it would therefore be advantageous to integratea read-out mechanism on the microscopic flexible structure. Furthermore,an integrated piezoresisitve sensor would provide a direct measure ofthe stress formation in the microscopic flexible structure. At present,very few experiments have been carried out on biological systems, whichnormally implies a liquid environment, and microscopic flexiblestructures with integrated read-out have rarely been applied.Furthermore, the majority of the micro-cantilever-based experimentscarried out until now have used micro-cantilevers developed for atomicforce microscopy. Such micro-cantilevers are not necessarily optimallydesigned for biochemical sensing.

[0107] The microscopic flexible structure-based sensors have a hugepotential, especially in the field of biochemical analysis. Thedetection technique can be used to construct smarter and simplerbiochemical detectors, but it should also allow novel studies of singlemolecular interactions due to the extremely high mechanical sensitivityof micro-mechanical structures.

[0108] According to the present invention, the microscopic flexiblestructure-based biochemical sensor is fully integrated in amicro-channel suitable for liquid flow measurements and the device ispreferably integrated with a micro-liquid handling system.

[0109] In a preferred embodiment of the present invention the sensorincludes:

[0110] 1) A supporting body made in silicon in which micro-channels areetched. The width of each channel is 100-500 μm and the depth is on theorder of 100 μm. The length of the channel is on the order of mm.

[0111] 2) Micro-cantilevers which extend partially across the width of amicro-channel. The micro-cantilevers are attached to the sidewall of thechannel. The micro-cantilevers are typically rectangular and areapproximately 50 μm wide, 200 μm long and 1 μm thick. Themicro-cantilevers are fabricated in silicon, silicon oxide and siliconnitride.

[0112] 3) An integrated detection system to measure changes in themechanical properties of the micro-cantilever. This system preferablycomprise piezoresistive elements on adjacent micro-cantilevers connectedwith similar resistive elements on the supporting body in order to forma Wheatstone bridge for accurate measurement of resistance changes inthe piezoresistor. The piezoresistors are placed on the top of themicro-cantilevers and the supporting body and they are fullyencapsulated in dielectric layers such as silicon oxide and siliconnitride.

[0113] 4) Electronic feed-throughs which ensure electrical contact tothe piezoresistive elements. The electrical wires are placed on top ofthe supporting body and the wire material is metal or highly dopedsilicon. The wires have a width of 100 μm, a thickness of approximately1 μm and a length on the order of mm.

[0114] 5) A spacer layer, which has to fully encapsulate the electricalwiring, so that liquid is not entering and short circuiting theelectrical connections. The spacer layer has a thickness of 100 μm sothat there is a clearance below and above the micro-cantilevers for theliquid to flow freely in the channel. The cover plate is fabricated in aUV curable polymer.

[0115] 6) A cover plate placed on top of the spacer layer. The coverplate has to form a hermetic sealing of the channel and is fabricated ina UV curable polymer and bonded to the spacer layer by a thermaltreatment. The cover plate has a thickness of approximately 100 μm.

[0116] For specific applications the sensor might further comprise:

[0117] 7) An integrated actuator mechanism which can be used to drivethe micro-cantilever at its resonance frequency or to induce a staticbending of the micro-cantilever. The micro-cantilever is actuated byeither electromagnetic/electrostatic forces or by integrating apiezoelectric layer or a heater element on the micro-cantilever. Forelectromagnetic/electrostatic actuation the micro-cantilever has to becoated with a conducting/magnetic material and externally exited by anelectric/magnetic field.

[0118] 8) A reference electrode for electrochemical measurements. Theelectrode must be in contact with the liquid and can be inserted throughthe cover plate.

[0119] Other realisations can involve different materials. The spacerlayer and cover plate can be fabricated in glass which is bonded to thesilicon support body. Micro-cantilever and supporting body can befabricated in polymer materials and the channels can be formed byembossing or injection moulding.

[0120] Other realisations can involve different detection techniques,such as external optical detection through the cover plate or integratedoptical systems where a displacement of the micro-cantilever modifiesthe transmission of an optical waveguide placed on or near themicro-cantilever. Other integrated detection principles could bepiezoelectric or capacitive. For piezoelectric detection of themicro-cantilever deflection a piezoelectric film is placed on themicro-cantilever, and for capacitive measurements the micro-cantileveris coated by a conducting film and a counter electrode is placed belowor above the micro-cantilever.

[0121] Other realisations can involve recessed micro-cantilevers (FIG.2), so that there is no need to form a spacer layer, or so that thecover plate can be eliminated, relying instead on capillary flow toguide fluids through channels. Furthermore, recessed micro-cantileverscan be used to perform measurements at different heights in the liquid.Moreover, the micro-cantilevers can be placed perpendicular to theliquid flow and micro-cantilevers can be placed on either side of thechannel.

[0122] On each micro-cantilever one piezoresistive element is placed. Byconnecting two micro-cantilevers and two resistors on the supportingbody in a Wheatstone bridge (FIG. 3) it is possible to perform a commonmode rejection of noise in the system. One micro-cantilever then servesas a reference micro-cantilever whereas the other is used to detect aspecific biochemical reaction. A reference measurement is crucial inliquid where turbulence and thermal drift have a significant influenceon the measurement.

[0123] Other realisations can involve triangular shapedmicro-cantilevers with piezoresistors placed on each of the two armsforming the triangular micro-cantilever (FIG. 4). This will enable thetorsion as well as the vertical deflection of the micro-cantilever to bedetected.

[0124] Other realisations can involve micro-bridges and micro-membranesinstead of micro-cantilevers.

[0125] In a second embodiment of the present invention, a completemicro-cantilever, micro-bridge or micro-membrane transducer systemcomprises the microstructure described in the above preferred embodimentof the invention, as well as:

[0126] 1) External electrical connections to the micro-cantilever,micro-bridge or micro-membrane system, to apply a controlled voltage tothe piezoresistive elements placed in Wheatstone bridges and to monitorthe electrical output from the piezoresistors.

[0127] 2) Voltage sources amplifiers and voltmeters to detect changes inthe piezoresistors due to a micro-cantilever bending.

[0128] 3) AC voltage source to apply AC signals to the piezoresistorsfor actuation or resonance detection.

[0129] 4) External fluidic connection to the micro-channels to pumpfluids in and out of the micro-channels.

[0130] Fabrication

[0131] The said sensor fully integrated in a micro-channel is fabricatedby use of micro-machining. This technique allows dimensions in themicrometer regime and high reproducibility. For the fabrication of amicro-bridge or a micro-cantilever sensor the fabrication is exactly thesame and only the design differs. In the following examples theresistors are defined in poly-crystalline silicon. By using asilicon-on-insulator wafer the resistors can be defined insingle-crystalline silicon which exhibits higher signal-to noise ratio.

EXAMPEL 1 Micro-Cantilever-Based Sensor

[0132] In the following, the fabrication of a micro-cantilever-basedsensor is described. The micro-cantilever consists of 5 layers, whereone of the layers serves as the piezoresistor. The sensor could also beformed with only three layers: A layer defining the piezoresistor and alayer on both sides of the resistor for the encapsulation.

[0133] The starting material is a 500 μm thick single side polished<100> silicon wafer. A 100-1000 nm thick thermal oxide is grown in orderto form an etch-stop layer for the later micro-cantilever releasing andchannel etch process. FIGS. 5.a-5.l show, in a side view, a schematicillustration of the process. A low pressure chemical vapour deposition(LPCVD) poly-silicon layer, 300-800 nm thick, is deposited on top of theoxide, succeeded by the growth of 500-1000 nm thick oxide for thefabrication of stress compensated micro-cantilevers (FIGS. 5.b-c).

[0134] A 200-350 nm LPCVD poly-silicon layer is deposited on top of theoxide. This layer defines the piezoresistive elements (piezoresistors)(FIG. 5.d). Boron is implanted in the poly-silicon to obtainapproximately a doping concentration of about 3·10¹⁹ cm⁻³. At thisdoping level a high gauge factor (K=30) and a low TCR (temperaturecoefficient of resistance) value (FIG. 5.e) are obtained.

[0135] A photo resist layer is spun on top of the wafer, and theresistor pattern is transferred to the resist by photolithography. Theresistors are then anisotropically etched in the poly-silicon byreactive ion etching (RIE) in order to obtain well-defined resistordimensions (FIG. 5.f).

[0136] Micro-cantilever and channel are then defined by a secondphotolithography step. The oxide/silicon/oxide layer can then be etchedby either (FIG. 5.g):

[0137] a) 1. Hydrofluoric acid (HF) etching of the top oxide layer

[0138] 2. Anisotropic RIE of the silicon

[0139] 3. HF etching of the bottom oxide layer

[0140] or

[0141] b) 1. Anisotropic RIE of the oxide

[0142] 2. Anisotropic RIE of the silicon

[0143] 3. Anisotropic RIE of the oxide

[0144] In order to encapsulate the resistors and to protect themicro-cantilever sidewalls when KOH/RIE etching of the channel, a 50-200nm thick thermal oxide is grown on top of the resistor (FIG. 5.h).Hereafter, a 20-100 nm thick LPCVD nitride is deposited to be used as anetch mask, but also as a diffusion barrier (FIG. 5.i).

[0145] For the fabrication of contact holes through the nitride/oxidelayer, a thin resist is spun on top of the wafer. The contact-hole maskis transferred to the resist by photolithography. The nitride is etchedby RIE and the oxide is etched by HF. The nitride/oxide in the channelsare also removed (FIG. 5.j)

[0146] Metal for electrical connections, typically chromium/gold oraluminium are deposited by lift-off technique. This is done by spinninga thin layer of resist on top of the wafer. The metal wire mask istransferred to the resist by photolithography. The metal is evaporatedon top of the wafer and finally the resist is stripped in acetoneleaving the metal wires on top of the support structure(FIG. 5.k).

[0147] For the use of metal as an immobilisation layer on themicro-cantilever, a metal layer is deposited on top of themicro-cantilever also by lift-off (FIG. 5.l). By depositingmicro-cantilever metal in a second lift-off step, it is possible to useother metals and metal thicknesses than used for the electricalconnections. Another metal layer can be deposited on the referencemicro-cantilever in order to make the two micro-cantilevers as identicalas possible

[0148] A schematic top view of the micro-cantilever-based biochemicalsensor at this point in the fabrication is shown in FIG. 6. The top ofthe channel structure is coated by nitride 1 and the sides and thebottom of the etched channel is silicon 2. The micro-cantilever iscoated by an optional metal layer for immobilisation of molecules 3, andthe micro-cantilever has a piezoresistor integrated 4. The piezoresistorhas two contact pads 5 to which metal wires have been connected.

[0149] In order to integrate the sensor in a closed channel, a top partof the channel is required. The upper part of the channel can befabricated by two different principles:

[0150] 1. The channel can be fabricated by depositing a spacer layer inpolymer, which actually defines the sidewalls of the top part of thechannel. A cover lid is then bonded or glued to the polymer.

[0151] 2. The upper part of the channel is etched in a glass or siliconwafer, which is bonded or glued to the micro-cantilever wafer.

[0152] The two fabrication procedures are described below:

[0153] Principle number 1 can be split out in two fabricationprocedures:

[0154] 1a): Closed Reactive Ion Etched Channel with Polymer Spacer:

[0155] A 30-100 μm thick photosensitive polymer layer is spun on the topside of the wafer seen in FIG. 5.m. The spacer mask is transferred tothe photosensitive polymer by photolithography, see FIG. 7.a

[0156] It is now possible to etch the channel and release themicro-cantilever by isotropic RIE using the metal on themicro-cantilever and the photosensitive polymer as etch masks. The depthof the channel is between 30-100 μm (FIG. 7.b).

[0157] The oxide below the micro-cantilever is etched by HF, yielding astress compensated micro-cantilever (FIG. 7.c).

[0158] Finally, a top plate consisting of silicon, Pyrex, polymer or anycombination of these is sealed to the photosensitive polymer wallseither by gluing or by local heating of the top of the photosensitivepolymer layer. The melted polymer will form a bond to the top plate(FIG. 7.d).

[0159] 1b) Closed KOH Etched Channel with Polymer Spacer

[0160] In order to fabricate a well-controlled channel the wafer isetched in KOH after the micro-cantilever has been defined and themicro-cantilever metal has been deposited. The metal and the nitride onthe micro-cantilever act as etch masks in this process. The KOH etch isfinished when the micro-cantilever is released resulting in a channeldepth of 30-100 μm, see FIG. 8.a.

[0161] The oxide below the micro-cantilever is etched by HF, yielding astress compensated micro-cantilever (FIG. 8.b).

[0162] A 30-100 μm thick polymer layer is transferred to the top side ofthe wafer, defineing the spacer layer see FIG. 8.c.

[0163] Finally, a top plate consisting of silicon, Pyrex, polymer or anycombination of these is sealed to the photosensitive polymer wallseither by gluing or by local heating of the top of the photosensitivepolymer layer. The melted polymer will form a bond to the top plate(FIG. 8.d).

[0164] 2): Closed Channel by Bonding the Top and the Bottom Part of theChannel:

[0165] In order to fabricate a well-controlled channel the wafer isetched in KOH after the micro-cantilever metal has been deposited. Themetal and the nitride act as etch masks in this process. The KOH etch isfinished when the micro-cantilever is released resulting in a channeldepth of 30-100 μm, see FIG. 9.a.

[0166] The oxide below the micro-cantilever is etched in HF, yielding astress compensated micro-cantilever (FIG. 9.b).

[0167] A 20-200 nm thick LPCVD nitride layer is deposited on a 500 μmthick single side polished <100> silicon wafer in which the top part ofthe channel is to be defined (FIG. 9.c).

[0168] Thin resist is spun on the back side of the wafer, and a maskdefining the holes for contacting the metal wires is transferred byphotolithography. The pattern is transferred to the nitride by RIE (FIG.9.d). The exposed silicon areas are then etched in KOH. The KOH etch isstopped when the created micro-membranes have a thickness 30-100 μm(FIG. 9.e).

[0169] Thin resist is then spun on the front side of the wafer and amask defining the channel and holes for contacting the metal wires istransferred to the resist by photolithography. The pattern istransferred to the nitride by RIE (FIG. 9.f). The channel and thecontact-hole are then etched until the 30-100 μm micro-membrane isetched away at the contact hole, resulting in a channel depth of 30-100μm (FIG. 9.g).

[0170] Between 2-10 μm glass is evaporated on the front side of thewafer for the anodic bonding process (FIG. 9.h). Finally, the two wafersare bonded by anodic bonding (FIG. 8.i).

[0171] Instead of using KOH etch in order to fabricate the upper part ofthe channel and the contact holes, it is possible to use RIE instead. Itis also possible to use a Pyrex wafer instead of a silicon wafer. For aPyrex wafer HF is used to isotropically etch the channel and thecontact-holes.

EXAMPEL 2 Micro-Membrane-Based Sensor

[0172] For the fabrication of a micro-membrane-based sensor in achannel, the fabrication is also performed by micromaching. In contrastto the micro-cantilever or micro-bridge-based sensor, the micro-membraneis normally placed in the bottom of the channel. This design makes itpossible to contact the resistors from the backside. Nevertheless, infollowing example the resistors will be contacted from the same side asthe channel.

[0173] The first steps ( FIGS. 5.a.-5.f.)in the fabrication sequence isbasically the same as descriebed for the micro-cantilever ormicro-bridge based sensor.

[0174] After the resistors have been defined by RIE the resistors areencapsulated in a 50-200 nm thick thermal oxide. Hereafter, a 20-100 nmthick LPCVD nitride is deposited to be used as an etch mask, but also asa diffusion barrier (FIG. 10.a.)

[0175] For the fabrication of contact holes through the nitride/oxidelayer, a thin resist is spun on top of the wafer. The contact-hole maskis transferred to the resist by photolithography. The nitride is etchedby RIE and the oxide is etched by HF (FIG. 10.b.).

[0176] Metal for electrical connections, typically chromium/gold oraluminium are deposited by lift-off technique. This is done by spinninga thin layer of resist on top of the wafer. The metal wire mask istransferred to the resist by photolithography. The metal is evaporatedon top of the wafer and finally the resist is stripped in acetoneleaving the metal wires on top of the support structure(FIG. 10.c.).

[0177] For the use of metal as an immobilisation layer on themicro-cantilever, a metal layer is deposited on top of themicro-membrane also by lift-off (FIG. 10.d.). By depositingmicro-cantilever metal in a second lift-off step, it is possible to useother metals and metal thicknesses than used for the electricalconnections.

[0178] The micro-membrane is now defined by KOH etching from thebackside. First, a thin resist layer is spun on the backside of thewafer. The backside mask is the tranferred to the resist. Hereafter, thenitride/silicon/oxide sandwich is etched in RIE. The wafer is thenetched in KOH, where the oxide will act as a etchstop. (FIG. 10.e.)

[0179] The oxide is then removed in a HF etch (FIG. 10.f.)

[0180] A channel is now fabricated on top of the micro-membrane. Thiscan be fabricated by two different principles:

[0181] 1. The channel can be fabricated by depositing a spacer layer inpolymer, which actually defines the sidewalls of the top part of thechannel. A cover lid is then bonded or glued to the polymer.

[0182] 2. The upper part of the channel is etched in a glass or siliconwafer, which is bonded or glued to the micro-cantilever wafer.

[0183] The two fabrication procedures are described below:

[0184] 1) Spacer Layer in Polymer

[0185] A 30-100 μm thick photosensitive polymer layer is spun on the topside of the wafer. The spacer mask is transferred to the photosensitivepolymer by photolithography, see FIG. 10.g

[0186] Finally, a top plate consisting of silicon, Pyrex, polymer or anycombination of these is sealed to the photosensitive polymer wallseither by gluing or by local heating of the top of the photosensitivepolymer layer. The melted polymer will form a bond to the top plate(FIG. 10.h).

[0187] 2) Closed Channel by Bonding the Top Part to the Substrate

[0188] This method is exactly the same as described in the “Closedchannel by bonding the top and the bottom part of the channel” sectionin the fabrication sequence of a micro-cantilever-based sensor in achannel.

Applications of the Present Invention

[0189] In the following, examples of different applications of thepresent invention are listed and commented. The application of thepresent invention should however not be limited to the listed examples.

[0190] Laminated Flow:

[0191] Adjacent or very closely spaced micro-cantilevers can be exposedto different chemical environments at the same time by (FIG. 11)

[0192] 1) Laminating the fluid flow vertically in the micro-channel intotwo or more streams, so that micro-cantilevers on opposing sides of themicro-channel are immersed in different fluids.

[0193] 2) Laminating the fluid flow horizontally in the micro-channel,so that micro-cantilevers recessed to different levels in themicro-channel are immersed in different fluids.

[0194] 3) Laminating the fluid flow either horizontally or verticallyand moving the micro-cantilevers through the different fluids byactuating the micro-cantilevers.

[0195] In this way, micro-cantilever signals from different fluidenvironments can be compared. Moreover the technology can be used forcoating narrowly spaced micro-cantilevers with different chemicalsubstances. Examples on both aspects will be described below.

[0196] Functionalisation:

[0197] Functionalising the micro-cantilevers can be performed usingconventional immobilisation chemistry, which easily applies to themicro-cantilever materials. However, for the closely spacedmicro-cantilevers in micro-channels new technologies for applying thedifferent coatings are needed. The functionalisation of narrowly spacedmicro-cantilevers can be performed by one or more of the technologiesdescribed below:

[0198] 1) In the micro-fabrication of the device, the micro-cantileverscan be coated with different thin film layers which are compatible withthe fabrication process. The thin films can be metal, silicon anddielectric layers. The different thin films can then be used to bindmolecules which have a specific binding to a specific thin film.

[0199] 2) The molecules to be attached on the micro-cantilever surfacecan be synthesised with a photo activated binding site. Molecules arethen attached to the micro-cantilever surface by placing themicro-cantilever in a liquid solution with the coating molecules andexposing the micro-cantilever to UV light. The UV light induces thecreation of a bond between the micro-cantilever surface and molecules.This coating can be performed in the channel after it has been closed,by injecting different coating molecules in the channel and illuminatingthe micro-cantilevers individually through the cover plate. By scanninga laser across the device small well-defined areas can be coated withspecific coatings. Between each coating the system must be rinsed and anew coating solution injected in the channels.

[0200] 3) Using an inkjet printer principle small droplets of liquid canbe delivered. These systems are commercially available for DNA chipfabrication. Such a liquid delivery system can be used to spray dropletsof different liquids on closely spaced micro-cantilevers. The delivereddroplets typically have a diameter of 100 μm. This coating techniquemust be performed before the channel is sealed.

[0201] 4) When the channels are sealed, laminated flow can be used tocoat narrowly spaced micro-cantilevers by having two or more laminatedflows in the system. Micro-cantilevers placed in different heightsand/or on different sides of the channel will thus be immersed indifferent liquids. After coating, the micro-channels can be flushed withother fluids to remove the residual coating material. By repeating thetechnique, several layers of coating can be added to themicro-cantilever. In order to bind molecules to only one side of themicro-cantilever photoimmobilisation or pre-deposited thin films can beused.

[0202] 5) Selective coating can be performed by laminating two or morestreams in the micro-channel and placing the micro-cantilever in one ofthe streams by a static bending. Moreover, a controlled movement of themicro-cantilever through separated laminated streams can be used to coatthe micro-cantilever with multiple layers such asglutaraldehyde-avidin-biotin.

[0203] 6) Selective and reversible coating of the micro-cantilever, withfor example metalloproteins, can be acheived electrochemically. Aconducting layer on the micro-cantilever can be used as the workingelectrode. The counter electrode might be an integrated part of thesystem. Also it is often desirable to include a reference electrode forcontrol of the applied potential.

[0204] Reference Micro-Cantilever and Reference Measurement:

[0205] To minimise the effect of turbulence and thermal drift in thesystem, a reference micro-cantilever can be implemented. The referencemicro-cantilever is placed close to the measurement micro-cantilever andin the same measurement environment. However, the referencemicro-cantilever is not coated with a detector film. The referencemicro-cantilever might be coated with another film which does not act asa detector or which detects a second substance. By subtracting thereference signal from the measurement signal most background noise canbe eliminated, see FIG. 12.

[0206] For most biochemical applications it is important to perform areference measurement in a reference liquid. Often it is theincrease/decrease in the concentration of a specific molecule which isof interest. For such relative measurements a reference liquid isnecessary. The micro-cantilever placed in the reference solution shouldbe identical to the measurement micro-cantilever in the measurementsolution, see FIG. 13. The measurement solution and the referencesolution can be investigated in the same channel at the same time bylaminating the flow and let the two streams run in parallel.Micro-cantilevers placed on either side of the channel will measure thereaction in two different fluids. Quasi-simultaneous measurements inanalytes and in reference solutions can be performed by moving themicro-cantilever through the two liquids.

[0207] Diffusion Measurements in Added Layers:

[0208] Molecules entering the detector films on the micro-cantileverchange the stress of the film, which results in a micro-cantileverbending. For example, diffusion in cell micro-membranes can beinvestigated and the activity of specific micro-membrane channels whichare regulated by voltage or by the binding of another molecule can beinvestigated.

[0209] Preliminary experiments on the diffusion of alcohol in polymershave been performed using micro-cantilevers with piezoresistiveread-out. One of two micro-cantilevers integrated in a Wheatstone bridgeis coated with a UV sensitive resist in which the stress is changed whensubjected to alcohol. FIG. 14 shows micro-cantilevers placed in a smallopen liquid container 7 with DI water. Liquid alcohol is injected 8 andthe output voltage from the Wheatstone bridge is recorded 9 as afunction of time. The output voltage from the Wheatstone bridge reflectsthe difference in the deflection of the two micro-cantilevers.

[0210] The micro-cantilever response to three different amounts ofEthanol is shown in FIG. 15. The arrows indicate the times at which newalcohol is placed on the surface of the water, close to themicro-cantilever. It is clearly seen, how the micro-cantilever respondsimmediately to the alcohol after which the signal decreases as thealcohol is diluted in the water and evaporated from the surface. Themagnitude of the signal reflects the amount of injected alcohol. Thusthe diffusion of alcohol into the polymer causes the stress in thepolymer to change. The process is reversible and when the alcohol leavesthe film, the micro-cantilever returns to the start position. Themechanism can be used to construct a sensor for measuring alcoholconcentrations in liquid.

[0211] The time dependent micro-cantilever response can also be used toinvestigate the dynamics of layer formation on the micro-cantileversurface. For example the formation of self-assembled monolayers can beinvestigated.

[0212] Conformal Changes of Protein Layers:

[0213] Conformal changes of proteins adsorbed on a micro-cantilever willgive rise to a change in resonance frequency and stress of themicro-cantilever. Hereby it is possible to study the conformal changesof proteins caused by external parameters such as pH-value,ion-concentration and temperature. For example the metalloprotein azurinadsorbed on gold is know to undergo conformational changes whensubjected to different pH-values. How azurin binds to gold, and how thebinding is changed when the pH-value is changed is not well understood,and the micro-cantilever-based measurements can give additionalinformation on the binding properties. Many active enzyme functions alsoresults in stress changes. Thereby enzyme activity levels in differentenvironments can be investigated.

[0214] Gene Detection:

[0215] One of the major applications of the invention is the detectionof multiple disease-associated genes. Single stranded DNA from thedisease-associated genes is attached to micro-cantilevers by one of thecoating technologies described above using conventional bindingchemistry. Narrowly spaced micro-cantilevers placed in one channel canbe coated with DNA sequences from different genes. A treated bloodsample consisting of single stranded DNA is then flushed through thesystem. If one of the disease-associated genes is present in the sampleit will bind specifically to the corresponding DNA string attached tothe micro-cantilever. DNA strings, which have been non-specificallybounded can be detached by a heat treatment. The specific binding willresult in a surface stress change as well as in a resonance change ofthe micro-cantilever. Hereby it is possible to perform a screening ofseveral genes simultaneously. The method could also apply to DNAsequencing.

[0216] Antigen-Antibody Reaction:

[0217] The idea of screening for specific genes can be expanded to thedetection of different antibodies. For this application closely spacedmicro-cantilevers are coated with different antigens, using conventionalbinding chemistries. Antibodies bind specifically to antigens, wherebyit is possible to screen for different antibodies in a blood sample.

[0218] Electrochemistry:

[0219] Applying a conducting layer on the micro-cantilever and areference electrode in the channel it is possible to performelectrodeposition and electrochemistry on layers on a micro-cantileversurface. For example in can be investigated how the stress in layers ofmettaloproteins such as azurin and yeast cytochrom c respond todifferent potentials. Furthermore redox-processes might be monitored.Moreover, the adsorption and desorption of electrodepositable moleculescan be investigated.

1. A sensor for detecting the presence of a substance in a fluid, saidsensor comprising: means for handling the fluid, said handling meanscomprising an interaction chamber of micrometer dimensions, an inlet andan outlet, a first flexible member having a surface, said surfaceholding a substance, wherein the surface holding the substance is atleast partly positioned inside the interaction chamber so that at leastpart of the substance is exposed to the fluid, and means for detecting amechanical parameter associated with the first flexible member, saidmechanical parameter being related to the presence of the substance inthe fluid.
 2. A sensor according to claim 1, wherein the first flexiblemember comprises a cantilever having a first and a second end, whereinthe first end is attached to the interaction chamber.
 3. A sensoraccording to claim 1, wherein the first flexible member comprises abridge having a first and a second end, wherein the first and secondends are attached to the interaction chamber.
 4. A sensor according toclaim 1, wherein the first flexible member forms part of a boundarydefining the interaction chamber.
 5. A sensor according to claim 1,wherein the detecting means for detecting the mechanical parameterassociated with the first flexible member comprises a piezoresistiveelement, said piezoresistive element being an integral part of the firstflexible member.
 6. A sensor according to claim 1, wherein the detectingmeans for detecting the mechanical parameter associated with the firstflexible member comprises a laser, an optical element and a positionsensitive photo detector.
 7. A sensor according to claim 6, wherein thepiezoresistive element forms part of a balanced bridge, such as aWheatstone bridge.
 8. A sensor according to claim 1 further comprisingan actuator for moving the flexible member relative to the interactionchamber.
 9. A sensor according to claim 8, wherein the actuatorcomprises a piezoelectric element.
 10. A sensor according to claim 8,wherein the actuator comprises means for providing an electrostaticinduced movement of the flexible member.
 11. A sensor according to claim8, wherein the actuator comprises means for providing a magnetic inducedmovement of the flexible member.
 12. A sensor according to claim 8,wherein the actuator comprises means for providing a thermal inducedmovement of the flexible member.
 13. A sensor according to claim 1,wherein the handling means is fabricated in a material selected from thegroup consisting of metals, glasses, polymers or semiconductormaterials, such as silicon.
 14. A sensor according to claim 1, whereinthe substance being held by the surface of the first flexible member isselected from the group consisting of metals, polymers, biochemicalmolecules or micro-biochemical structures.
 15. A sensor according toclaim 1, further comprising a second flexible member being at leastpartly positioned inside the interaction chamber so that at least partof the second flexible member is exposed to the fluid, and means fordetecting a mechanical parameter associated with the second flexiblemember.
 16. A sensor according to claim 15, wherein the detecting meanscomprises a piezoresistive element, said piezoresistive element being anintegral part of the second flexible member, and wherein thepiezoresistive element forms part of a balanced bridge, such as aWheatstone bridge.
 17. A sensor for detecting the presence of asubstance in a fluid, said sensor comprising: means for handling thefluid, said handling means comprising an interaction chamber, an inletand an outlet, a first flexible member having a surface, said surfaceholding a substance, wherein the surface holding the substance is atleast partly positioned inside the interaction chamber so that at leastpart of the substance is exposed to the fluid, and means for detecting amechanical parameter associated with the first flexible member, saidmechanical parameter being related to the presence of the substance inthe fluid, wherein the detecting means form an integral part of thefirst flexible member.
 18. A sensor according to claim 17, wherein thefirst flexible member comprises a cantilever having a first and a secondend, wherein the first end is attached to the interaction chamber.
 19. Asensor according to claim 17, wherein the detecting means for detectingthe mechanical parameter associated with the first flexible membercomprises a piezoresistive element.
 20. A sensor according to claim 19,wherein the piezoresistive element forms part of a balanced bridge, suchas a Wheatstone bridge.
 21. A sensor according to claim 17, wherein theinteraction chamber is of micrometer dimensions.
 22. A sensor accordingto claim 18 further comprising an actuator for moving the second end ofthe cantilever relative to the interaction chamber.
 23. A sensoraccording to claim 22, wherein the actuator comprises a piezoelectricelement, said piezoelectric element being an integral part of thecantilever.
 24. A sensor according to claim 17, wherein the handlingmeans is fabricated in a material selected from the group consisting ofmetals, glasses, polymers or semiconductor materials, such as silicon.25. A sensor according to claim 17, wherein the substance being held bythe surface of the first flexible member is selected from the groupconsisting of metals, polymers, biochemical molecules ormicro-biochemical structures.
 26. A sensor according to claim 17,further comprising a second flexible member being at least partlypositioned inside the interaction chamber so that at least part of thesecond flexible member is exposed to the fluid, and means for detectinga mechanical parameter associated with the second flexible member.
 27. Asensor according to claim 26, wherein the detecting means comprises apiezoresistive element, said piezoresistive element being an integralpart of the second flexible member, and wherein the piezoresistiveelement forms part of a balanced bridge, such as a Wheatstone bridge.28. A sensor for detecting the presence of a substance in a fluid, saidsensor comprising: means for handling the fluid, said handling meanscomprising an interaction chamber, an inlet and an outlet, a firstflexible member having a surface, said surface holding a substance,wherein the surface holding the substance is at least partly positionedinside the interaction chamber so that at least part of the substance isexposed to the fluid, and wherein the first flexible member forms anintegral part of the handling means, and means for detecting amechanical parameter associated with the first flexible member, saidmechanical parameter being related to the presence of the substance inthe fluid.
 29. A sensor according to claim 28, wherein the firstflexible member comprises a cantilever having a first and a second end,wherein the first end is attached to the interaction chamber.
 30. Asensor according to claim 28, wherein the detecting means for detectingthe mechanical parameter associated with the first flexible membercomprises a piezoresistive element, said piezoresistive element being anintegral part of the first flexible member.
 31. A sensor according toclaim 30, wherein the piezoresistive element forms part of a balancedbridge, such as a Wheatstone bridge.
 32. A sensor according to claim 28,wherein the detecting means for detecting the mechanical parameterassociated with the first flexible member comprises a laser, an opticalelement and a position sensitive photo detector.
 33. A sensor accordingto claim 28 further comprising an actuator for moving the second end ofthe cantilever relative to the interaction chamber.
 34. A sensoraccording to claim 33, wherein the actuator comprises a piezoelectricelement, said piezoelectric element being an integral part of thecantilever.
 35. A sensor according to claim 28, wherein the handlingmeans is fabricated in a material selected from the group consisting ofmetals, glasses, polymers or semiconductor materials, such as silicon,and wherein the interaction chamber is of micrometer dimensions.
 36. Asensor according to claim 28, wherein the substance being held by thesurface of the first flexible member is selected from the groupconsisting of metals, polymers, biochemical molecules ormicro-biochemical structures.
 37. A sensor according to claim 28,further comprising a second flexible member being at least partlypositioned inside the interaction chamber so that at least part of thesecond flexible member is exposed to the fluid, and means for detectinga mechanical parameter associated with the second flexible member.
 38. Asensor according to claim 37, wherein the detecting means comprises apiezoresistive element, said piezoresistive element being an integralpart of the second flexible member, and wherein the piezoresistiveelement forms part of a balanced bridge, such as a Wheatstone bridge.39. A sensor for detecting the presence of a substance in a fluid, saidsensor comprising: means for handling the fluid, said handling meanscomprising an interaction chamber, an inlet and an outlet, a firstflexible member having a surface, said surface holding a substance,wherein the surface holding the substance is at least partly positionedinside the interaction chamber so that at least part of the substance isexposed to the fluid, and wherein fabrication of the first flexiblemember is part of fabrication of the handling means, means for detectinga mechanical parameter associated with the first flexible member, saidmechanical parameter being related to the presence of the substance inthe fluid.
 40. A sensor according to claim 39, wherein the firstflexible member comprises a cantilever having a first and a second end,wherein the first end is attached to the interaction chamber.
 41. Asensor according to claim 39, wherein the detecting means for detectingthe mechanical parameter associated with the first flexible membercomprises a piezoresistive element, said piezoresistive element being anintegral part of the first flexible member.
 42. A sensor according toclaim 40 further comprising an actuator for moving the second end of thecantilever relative to the interaction chamber.
 43. A sensor accordingto claim 39, wherein the handling means is fabricated in a materialselected from the group consisting of metals, glasses, polymers orsemiconductor materials, such as silicon, and wherein the interactionchamber is of micrometer dimensions.
 44. A sensor according to claim 39,wherein the substance being held by the surface of the first flexiblemember is selected from the group consisting of metals, polymers,biochemical molecules or micro-biochemical structures.
 45. A sensoraccording to claim 39, further comprising a second flexible member beingat least partly positioned inside the interaction chamber so that atleast part of the second flexible member is exposed to the fluid, andmeans for detecting a mechanical parameter associated with the secondflexible member.
 46. A sensor according to claim 45, wherein thedetecting means comprises a piezoresistive element, said piezoresistiveelement being an integral part of the second flexible member, andwherein the piezoresistive element forms part of a balanced bridge, suchas a Wheatstone bridge.
 47. A sensor for detecting the presence of afirst and a second substance in a fluid, said sensor comprising: meansfor handling the fluid, said handling means comprising an interactionchamber of micrometer dimensions, an inlet and an outlet, a firstflexible member having a surface, said surface holding a firstsubstance, wherein the surface holding the first substance is at leastpartly positioned inside the interaction chamber so that at least partof the first substance is exposed to the fluid, a second flexible memberhaving a surface, said surface holding a second substance, wherein thesurface holding the second substance is at least partly positionedinside the interaction chamber so that at least part of the secondsubstance is exposed to the fluid, a first detecting means for detectinga first mechanical parameter associated with the first flexible member,said first mechanical parameter being related to the presence of thefirst substance in the fluid, and a second detecting means for detectinga second mechanical parameter associated with the second flexiblemember, said second mechanical parameter being related to the presenceof the second substance in the fluid.
 48. A sensor according to claim47, wherein each of the first and second flexible members comprises acantilever having a first and a second end, wherein the first end isattached to the interaction chamber.
 49. A sensor according to claim 47,wherein each of the first and second flexible members comprises a bridgehaving a first and a second end, wherein the first and second ends areattached to the interaction chamber.
 50. A sensor according to claim 47,wherein each of the first and second flexible members forms part of aboundary defining the interaction chamber.
 51. A sensor according toclaim 47, wherein the first detecting means comprises a piezoresistiveelement, said piezoresistive element being an integral part of the firstflexible member.
 52. A sensor according to claim 47, wherein the seconddetecting means comprises a piezoresistive element, said piezoresistiveelement being an integral part of the second flexible member.
 53. Asensor according to claim 51, wherein each of the piezoresistiveelements forms part of a balanced bridge, such as a Wheatstone bridge.54. A sensor according to claim 47, wherein the detecting means fordetecting the first and second mechanical parameters associated with thefirst and second flexible member, respectively, comprises a laser, anoptical element and a position sensitive photo detector.
 55. A sensoraccording to claim 47 further comprising actuators for moving the firstand second flexible members relative to the interaction chamber.
 56. Asensor according to claim 55, wherein the actuators comprisepiezoelectric elements, said piezoelectric elements being an integralpart of the cantilevers.
 57. A sensor according to claim 55, wherein theactuators comprise means for providing an electrostatic induced movementof the second end of the cantilevers.
 58. A sensor according to claim55, wherein the actuators comprise means for providing a magneticinduced movement of the second end of the cantilevers.
 59. A sensoraccording to claim 55, wherein the actuators comprise means forproviding a thermal induced movement of the second end of thecantilevers.
 60. A sensor according to claim 47, wherein the handlingmeans is fabricated in a material selected from the group consisting ofmetals, glasses, polymers or semiconductor materials, such as silicon.61. A sensor according to claim 47, wherein the substances being held bythe surface of the first and second flexible members are selected fromthe group consisting of metals, polymers, biochemical molecules ormicro-biochemical structures.
 62. A sensor for detecting the presence ofa first and a second substance in a moving laminated fluid, saidlaminated fluid comprising, in a cross section perpendicular to adirection of movement, a first and a second region, said sensorcomprising: means for handling the laminated fluid, said handling meanscomprising an interaction chamber, an inlet and an outlet, a firstflexible member having a surface, said surface holding a firstsubstance, wherein the surface holding the first substance is at leastpartly positioned inside the interaction chamber so that at least partof the first substance is exposed to the first region of the laminatedfluid, a second flexible member having a surface, said surface holding asecond substance, wherein the surface holding the second substance is atleast partly positioned inside the interaction chamber so that at leastpart of the second substance is exposed to the second region of thelaminated fluid, means for detecting a first mechanical parameterassociated with the first flexible member, said first mechanicalparameter being related to the presence of the first substance in thefirst region of the fluid, and means for detecting a second mechanicalparameter associated with the second flexible member, said secondmechanical parameter being related to the presence of the secondsubstance in the second region of the fluid.
 63. A sensor according toclaim 62, wherein the detecting means for detecting the first mechanicalparameter associated with the first flexible member comprises apiezoresistive element, said piezoresistive element being an integralpart of the first flexible member.
 64. A sensor according to claim 63,wherein the detecting means for detecting the second mechanicalparameter associated with the second flexible member comprises apiezoresistive element, said piezoresistive element being an integralpart of the second flexible member.
 65. A sensor according to claim 62,wherein the detecting means for detecting the first and secondmechanical parameters associated with the first and second flexiblemember, respectively, comprises a laser, an optical element and aposition sensitive photo detector.
 66. A sensor according to claim 62,further comprising a first actuator for moving part of the firstflexible element relative to the handling means.
 67. A sensor accordingto claim 66, wherein the first actuator comprises a piezoelectricelement, said piezoelectric element being an integral part of the firstflexible member.
 68. A sensor according to claim 62, further comprisinga second actuator for moving part of the second flexible elementrelative to the handling means.
 69. A sensor according to claim 68,wherein the second actuator comprises a piezoelectric element, saidpiezoelectric element being an integral part of the second flexiblemember.
 70. A sensor according to claim 62, wherein the handling meansis fabricated in a material selected from the group consisting ofmetals, glasses, polymers or semiconductor materials, such as silicon.71. A sensor according to claim 62, wherein the substances being held bythe surface of the first and second flexible members are selected fromthe group consisting of metals, polymers, biochemical molecules ormicro-biochemical structures.